骨骼肌两型肌纤维体外培养条件的初探

发布时间：2018-01-03 03:46

本文关键词：骨骼肌两型肌纤维体外培养条件的初探　出处：《遵义医学院》2014年硕士论文　论文类型：学位论文

【摘要】：目的：通过分别对体外培养条件下的骨骼肌两型肌纤维细胞的标志蛋白MHC基因同功型的检测，以探索适合骨骼肌两型肌纤维细胞的培养条件，为进一步研究两型肌纤维细胞的区别及转化机制奠定基础。方法：实验以18只家兔作为研究对象，不拘雌雄,4-6周，体重500-1000g，每项指标检测各用3只。动物分四组：Ⅰ型和Ⅱ型骨骼肌组织组，Ⅰ型和Ⅱ型骨骼肌消化细胞组，Ⅰ型和Ⅱ型骨骼肌细胞常氧（1、3、7天）型和Ⅰ型和Ⅱ型骨骼肌细胞低氧（3%O2）培养组。取家兔后肢右侧半膜肌的固有半膜肌亚部和副半膜肌背侧亚部，采用胰蛋白酶一步消化法，用含15%胎牛血清的高糖DMEM作为细胞培养液进行常氧和低氧培养，两型肌纤维培养细胞分别在培养1天、3天、7天取材进行观察。最后用常氧及低氧培养3天的两型骨骼肌细胞做反转录聚合酶链式反应（Rt-PCR）鉴定和灰度值测定作对照，以分析骨骼肌两型肌纤维细胞mRNA标志蛋白MHC的表达变化。结果：1.骨骼肌组织检测结果：固有半膜肌表达MHCⅠ及少量的MHCⅡ型，副半膜肌表达MHCⅡa, MHCⅡb型。 2.消化后未经培养的细胞检测结果：固有半膜肌细胞表达MHCⅠ型、MHCⅡa和MHCⅡb型，副半膜肌表达MHCⅡa、 MHCⅡb型。 3.培养细胞检测结果：在常氧培养组固有半膜肌表达MHCⅡa、 MHCⅡb，，不表达MHCⅠ型；副半膜肌表达MHCⅡa, MHCⅡb型，不表达MHCⅠ型。在低氧培养组两型骨骼肌细胞均表达为MHCⅡa, MHCⅡb，不表达MHCⅠ型。 4.我们选取骨骼肌组织组、培养后第3天的常氧培养组和低氧培养组进行单因素方差分析统计，结果显示：Ⅰ型肌纤维细胞在常氧及低氧条件的MHCⅠ型基因表达与组织相比较存在明显差异（P＜0.05），而MHCⅡa、MHCⅡb和MHCⅡx型基因表达无明显差异(P＞0.05)。低氧组与常氧组相比，MHCⅠ、MHCⅡa较高，而MHCⅡb较低。Ⅱ型肌纤维细胞在常氧及低氧条件的基因MHCⅠ、MHCⅡa、MHCⅡb和MHCⅡx型表达与组织相比较无明显差异(P＞0.05)。低氧组与常氧组比较MHCⅠ、MHCⅡb较高，MHCⅡa较低。结论：1.含15%胎牛血清高糖DMEM适合骨骼肌细胞生长。 2.骨骼肌两型肌纤维在上述培养条件下均有由Ⅰ型纤维向Ⅱ型纤维转化的趋势。 3．低氧培养条件有促进Ⅰ型纤维向Ⅱ型纤维转化的作用。
[Abstract]:Objective: through detecting signs under the conditions of the skeletal muscle of type two muscle fibers in vitro protein MHC gene isoform, culture conditions to explore for skeletal muscle of type two muscle fiber cells, and lay the foundation for the further study of the mechanism of transformation and difference between two types of muscle fiber cells.
Methods: the experiment with 18 rabbits as the research object, both male and female, 4-6 weeks, weight 500-1000g, each index to detect the 3. The animal were divided into four groups: type I and type II skeletal muscle group, type I and type II skeletal muscle of the digestive cells group, type I and type II skeletal muscle cells often oxygen (1,3,7 days) and type I and type II skeletal muscle cell hypoxia (3%O2) group. The rabbit right hindlimb culture Semimembranous muscle dorsal inherent semimembranosus muscle compartment and vice semimembranosus sub department, step by trypsin digestion method, containing 15% fetal bovine serum glucose as DMEM cell culture fluid in normoxia and hypoxia, type two muscle fibers in cultured cells were cultured for 1 days, 3 days, 7 days were observed. In normoxia and hypoxia training 3 days of type two skeletal muscle cells do reverse transcription polymerase chain reaction (Rt-PCR) identification and gray value was measured as control. In order to analyze the skeletal muscle of type two muscle fibers Changes in the expression of mRNA marker protein MHC.
Results: the testing results of 1. skeletal muscle tissue: MHC I and a small amount of MHC type II expression inherent semimembranosus, expression of MHC II a vice semimembranosus, MHC type.
2. after digestion without cell culture test results: the expression of the inherent semimembranosus cell type MHC, a and MHC II MHC II B, MHC II a expression of vice semimembranosus, MHC type.
3. test results: cultured cells cultured in normoxic group inherent semimembranosus muscle expression of MHC II A, MHC II B, the expression of MHC type 1; MHC II a expression of vice semimembranosus, MHC type, MHC type. The expression of cultured type two skeletal muscle cells were expressed as MHC II A in hypoxia MHC, of B, the expression of MHC type.
We selected 4. groups of skeletal muscle tissue, cultured after third days of normoxia group and hypoxia group were analyzed by one-way ANOVA statistics, the results show: compared with the organization difference expression of MHC type I type I muscle cells under normoxia and hypoxia condition gene (P < 0.05), and MHC II A, B and MHC II MHC II type X gene expression had no significant difference (P > 0.05). Compared with normoxia group and hypoxia group, MHC I, MHC II and MHC B II a high, low. Gene MHC I type II muscle cells under normoxia and hypoxia conditions, MHC II a. MHC II B and MHC II X expression compared with tissue had no significant difference (P > 0.05). Hypoxia group and normoxia group MHC 1, MHC 2 B high MHC II A is low.
Conclusion: 1. high glucose DMEM containing 15% fetal bovine serum is suitable for the growth of skeletal muscle cells.
2. the two muscle fibers of skeletal muscle were transformed from type I fiber to type II fiber under the above culture conditions.
3. the condition of low oxygen culture can promote the conversion of type I fiber to type II fiber.

【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R336

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